Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene

摘要

AIM: To design a hammerhead ribozyme targeting humantelomerase reverse transcriptase (hTERT) and clone it's genefor future use in the study of tumor gene therapy.METHODS: Using the software RNAstructure, the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected. A hammerhead ribozymetargeting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted. The DNA encodingthe ribozyme was synthesized and cloned into pGEMEX-1and the sequence of the ribozyme gene was confirmed byDNA sequencing.RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme. The designed ribozymewas comprised of 22nt catalytic core and 17nt flankingsequence. Computer-aided prediction suggested that theribozyme and hTERT mRNA could cofold into a properconformation. Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION: This fundamental work of successfuldesigning and cloning of an anti-hTERT hammerheadribozyme has paved the way for further study of inhibitingtumor cell growth by cleaving hTERT mRNA with ribozyme.